Currently, selection of the embryos with the highest implantation potential during assisted reproductive technology (ART) procedures relies only on morphological criteria. A new method based on time-lapse imaging has been recently described for the acquisition of embryo morphokinetic data to help such selection (Meseguer et al., 2011; Herrero and Meseguer, 2013). Nevertheless, the subjective observation of embryo morphology to predict a successful pregnancy shows limitations (Guerif et al., 2007; Assou et al., 2008; Aydiner et al., 2010; Assou et al., 2010). Therefore, many recent works have focused on the identification of new non-invasive biomarkers based on the analysis of the oocyte microenvironment to improve the accuracy of embryo selection (Pearson, 2006; Assou et al., 2008; van Montfoort et al., 2008; Assou et al., 2010; Aydiner et al., 2010; Uyar et al., 2013). In some studies, follicular fluid (FF) components, which are derived from plasma or secreted from granulosa cells, were investigated as potential biomarkers (De Placido et al., 2006; Baka and Malamitsi-Puchner, 2006; Yanaihara et al., 2007; Estes et al., 2009; Revelli et al., 2009; Borowiecka et al., 2012; Lédée et al., 2013). Indeed, FF, which surrounds the oocyte, is involved in follicular maturation, oocyte growth and the gradual acquisition of developmental competence. Consequently, FF might represent a reliable source of oocyte and embryo outcome biomarkers that could be used as supplemental prognostic/diagnostic tools in ART (Mermillod et al., 1999; Mendoza et al., 2002; Sutton et al., 2003; Krisher, 2004; Angelucci et al., 2006; Baka and Malamitsi-Puchner, 2006).
Cell-free DNA (cfDNA) fragments can be detected in the bloodstream (Mandel and Métais, 1948; Swarup and Rajeswari, 2007) and result from apoptotic or necrotic processes. They are released via a passive or an active mechanism (Jahr et al., 2001; Stroun et al., 2001). Nuclear and mitochondrial DNA can be released passively in the blood from apoptotic or necrotic cells (Schwarzenbach et al., 2011) and are then phagocytized by macrophages in healthy individuals, in whom the basal cfDNA level remains low (Jiang et al., 106 2003; Pisetsky and Fairhurst, 2007). CfDNA can also be actively secreted by cells (Gahan et al., 2008), leading to an increase of cfDNA circulating level in some cancers and other serious disorders. For that reason, cfDNA is used as non-invasive diagnostic and/or prognostic biomarker for some cancers and other severe pathologies (Paci et al., 2009; Vlassov et al., 2010; Gao et al., 2010; Kamat et al., 2010; Schwarzenbach et al., 2011; Gahan, 2012; Chen et al., 2013; da Silva Filho et al., 2013). Similarly, the emergence of non-invasive prenatal testing, based on foetal cfDNA detection in the maternal blood, constitutes a promising approach in obstetrics and gynaecology (Wright and Burton, 2009, Liao et al., 2014). So far, no study has evaluated cfDNA content in ovarian follicles, although follicular atresia is the result of many apoptotic events in granulosa cells.
In the present invention, the inventors asked whether cfDNA could be detected in FF and whether its quantification could be used to develop an innovative prognostic test for embryo quality. To this aim, the inventors quantified cfDNA in the FF of individual pre-ovulatory follicles from patients undergoing conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The inventors then explored whether cfDNA level and integrity were related to the follicle size and hormonal content, the patients' clinical characteristics and IVF outcomes. The inventors demonstrate that cfDNA quantification in FF represent an innovative and non-invasive biomarker to improve embryo selection in IVF procedures.